CN108558990A - Structure, expression and its application of the cowpea sheding green mottled virus sample particle of cancer target peptide F3 modifications - Google Patents
Structure, expression and its application of the cowpea sheding green mottled virus sample particle of cancer target peptide F3 modifications Download PDFInfo
- Publication number
- CN108558990A CN108558990A CN201810396256.7A CN201810396256A CN108558990A CN 108558990 A CN108558990 A CN 108558990A CN 201810396256 A CN201810396256 A CN 201810396256A CN 108558990 A CN108558990 A CN 108558990A
- Authority
- CN
- China
- Prior art keywords
- ccmv
- virus
- target peptide
- cancer target
- particles
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/14011—Bromoviridae
- C12N2770/14023—Virus like particles [VLP]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Wood Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides the structure of the cowpea sheding green mottled virus sample particle of cancer target peptide F3 modifications and its applications.By the way that cancer target peptide F3 to be inserted into the CCMV capsid proteins CP for knocking out 1 26 amino acid residue of amino terminal, structure obtains F3 CCMV virus-like particles.Using F3 CCMV virus-like particles as targeted drug delivery vehicles, it contains nir dye IR780 iodide, prepares F3 CCMV IR780 nano particles, it being capable of target recognition of tumor cell, tumour cell is killed using photo-thermal effect, plays the effect of biological immune targeted therapy.
Description
Technical field：
The present invention relates to genetic engineering field, be a kind of plant virus sample particle modification, expression and preparation method and its
Using structure, expression and its application of the cowpea sheding green mottled virus sample particle of specially cancer target peptide F3 modifications.
Background technology：
Virus-like particle (Virus-like particles, VLPs) is free from the hollow shell structure of viral nucleic acid, in form
It is similar to natural virion in structure, there is very strong immunogenicity and biological activity.Since VLPs is without containing virus
Inhereditary material, no infectivity, some have been used as vaccine to be applied successfully.Many virus structural proteins all have self assembly at VLPs
Ability, can acquisition be generated by the heterologous expression system of non-former host.
The nanoparticle vector built based on VLPs is had the advantages such as good biocompatibility, specific targeting be strong, is
Ideal delivery system carrier.Virus nanoparticles inner space can contain the small molecules such as polypeptide, drug or tracer
The macromolecular substances such as substance and nucleic acid, albumen or nanoparticle.And existed by the means such as chemical modification and genetic engineering transformation
The surfaces VLPs are fitted into various molecules of interest, including folic acid, polyethylene glycol, aptamers, polypeptide and nanoparticle etc., and clothing also can be changed
The amino acid sequence and type of glutelin, targeting, drugloading rate and encapsulation rate etc. to improve carrier.
Cowpea sheding green mottled virus (Cowpea chlorotic mottle virus, CCMV) is studied as plant virus
Type sepecies, discussion is had attracted much attention in field of nano biotechnology, by as nano-reactor, nanometer developer, drug/base
Because delivery vehicles etc. elaborate.CCMV virus-like particles are regular dodecahedron structure, by 90 capsid protein (Capsid
Protein, CP) dimer composition, it is symmetrically arranged with Triangulation number T=3, being carried out at different pH and ionic strength can
Inverse self depolymerization/package assembly transfer process can also form 28nm (T=3) or 18nm (pseudo T=2) VLPs.Root
According to this characteristic, Cornelissen etc. contains anionic polymer such as poly styrene sulfonate (PSS), poly-ferrocene with CCMV
Base silane (PFS), four sulfonato phthalocyanine zinc (ZnPc) and phthalocyanine (Pc) dendrimers form monodisperse or the T=1 of polydispersion,
2,3VLPs, even capsid protein-dendrimers biology heterozygosis nano particle；These nano particles show good
Photodynamics and photo-thermal therapy effect are targeted, 620-660nm Hg lamp irradiations is used after being such as incubated with macrophage, irradiates part cell
Death rate 92%-95%, irradiated portion cell survival are good.Douglas and Young etc. modifies CCMV clothing by genetic engineering
42 lysine is substituted for arginine and is cysteine for orthomutation by 102 and 130 serines by glutelin,
It selectively reacts to form covalent linkage with the sulfydryl of cysteine with the photosensitizer containing ruthenium with iodacetyl amido, finally
Biotin is connected to capsid protein surface by acylation reaction, the CCMV after modification can be used as a nanometer developer.Animal toxicology
Experiment shows lung, kidney and liver aggregation in Mice Body after CCMV intravenous injections, and body can be discharged in major part VLPs in 24 hours
Outside, biodegradable, without infectious and additional toxicity.
Cancer target peptide F3 is to be located at (the Human high-mobility group of mankind's high mobility group protein 2
Protein 2, HMGN2) aminoterminal 31 amino acid residues, sent out by display technique of bacteriophage equal to 2002 by Porkka
It is existing, there is Cell permeable.F3 peptides can identify the paranuclein in conjunction with tumour cell and endothelial cells in tumor neogenetic blood vessels surface
(Nucleolin), and paranuclein in normal cell only expressed in core.By paranuclein, F3 peptides are selectively thin with tumour
Born of the same parents and tumor vascular endothelial cell combine, the compact texture without penetrating solid tumor.Reddy etc. is prepared for the targeting of F3 peptides
Organic nanometer granule polymer, package developer (iron oxide or fluorescence) and photosensitizer Photofrin, in vitro study show that this is received
Rice grain can be combined, be internalized by and be transported in neoplastic cell nuclei, and tumour cell, In vivo NMR imaging are killed in photoactivation
(MRI) display nano particle may act on mouse brain glioma cell line 9L, and the photosensitizer than not wrapping up dramatically increases mouse
Time-to-live and therapeutic effect.Using F3 peptides as cancer target site modification virus sample particle surface, there is not been reported.IR780
Iodide belong to nir dye, have than the indocyanine green of clinical application (ICG) higher, more stable fluorescence intensity, can use
In the photo-thermal therapy PTT of laser irradiation, but its lipophilicity limits its application.IR780 is by human transferrin and the white egg of serum
It is white to contain acquisition nano particle, increase solubility, reduces cytotoxicity, and show encouraging light thermal property.
Currently, the research of cancer target viral vectors is mostly based on animal virus sample particle, the targeting peptides being related to are main
Including RGD, VEGF etc., the drug conveying carrier based on plant virus sample particle and effective expression and application how are established, is this
Technical problems to be solved by the inivention.
Invention content：
It is an object of the invention to cancer target peptide F3 is introduced the CCMV clothing that amino terminal knocks out partial amino-acid residue
In glutelin CP (Δ N26), gene fusion construct F3-CP (Δ N26), induced expression, purified fusion in Bichi yeast system
Albumen is obtained the F3-CCMV virus-like particles of cancer target peptide modification, is contained with it using the self assembly characteristic of capsid protein
Nir dye IR780 iodide research and develop the Nano medication delivery vehicles of cancer target.
Structure, expression and its application of the cowpea sheding green mottled virus sample particle of the cancer target peptide F3 modification of the present invention be
It is achieved through the following technical solutions：
A kind of cowpea sheding green mottled virus sample particle of cancer target peptide modification, CCMV capsid proteins CP knock out amino terminal
1-26 amino acid residues are inserted into cancer target peptide F3, build F3-CP fusions, obtain F3-CCMV virus-like particles.
The conjunction of fusion F3-CP (Δ N26) is carried out using dual asymmetric PCR and Overlap extension PCR two-step PCR
At wherein F3 genes are inserted into CCMV CP (Δ N26) gene that the ends N- knock out 1-26 amino acid residues.
The expression of the cowpea sheding green mottled virus sample particle of the cancer target peptide modification, the specific steps are：
(1) structure of fusion F3-CP (Δ N26)
Fusion is carried out using dual asymmetric PCR (DA-PCR) and Overlap extension PCR (OE-PCR) two-step PCR
The synthesis of F3-CP (Δ N26), wherein F3 genes are inserted into CCMV CP (Δ N26) base that the ends N- knock out 1-26 amino acid residues
Because in.
(2) structure of recombinant expression plasmid pPICZ A-F3-CP (Δ N26)
Fusion F3-CP (Δ N26) is inserted on Pichia pastoris shuttle vector pPICZ A by digestion, connection, is obtained
Obtain recombinant expression plasmid pPICZ A-F3-CP (Δ N26).
(3) conversion, screening of Pichia pastoris
Pichia pastoris, culture, screening positive transformants are converted after recombinant plasmid pPICZ A-F3-CP (Δ N26) is linearized
Strain.
(4) induced expression of recombinant protein F3-CP
Methanol induction is carried out to Pichia pastoris positive transformants, thalline were collected by centrifugation, passes through proteins gel electrophoresis after broken wall
(SDS-PAGE) expression of F3-CP is detected.
(5) purifying of recombinant protein F3-CP
Purification of recombinant proteins F3-CP is resuspended to assembling buffer solution to obtain F3-CCMV virus-like particles.
(6) identification of F3-CCMV virus-like particles
The F3-CCMV virus-like particles of acquisition are detected into its form and grain size with transmission electron microscope TEM and dynamic light scattering DLS
Size.
Pichia pastoris described in step (3) is Pichia pastoris GS115, and condition of culture is to contain bleomycin Zeocin's
YPD tablets (1% yeast extract, 2% peptone, 2% glucose, 2% agar powder).
Polyethylene glycol PEG sedimentations and cesium chloride density gradient centrifugation purification of recombinant proteins F3-CP are utilized in step (5).
The cowpea sheding green mottled virus sample particle of the cancer target peptide modification is identifying and is killing MCF-7 tumour cells
In application.
The application, includes the following steps：
(1) preparation of F3-CCMV-IR780 nano particles
Using reversible self depolymerization/package assembly transfer characteristics of CCMV, IR780 is contained with F3-CCMV virus-like particles
Iodide detach through ultrafiltration centrifugation, FPLC, prepare F3-CCMV-IR780 nano particles；
(2) the cell in vitro intake of F3-CCMV-IR780 nano particles
Using human breast carcinoma cell lines MCF-7 as indicator cells, F3-CCMV-IR780 nano particles are to the external of tumour cell
Intake and targeting binding ability；
(3) cell viability detects
The near-infrared effect of IR780 is induced by laser irradiation, F3-CCMV-IR780 nano particles are killed using photo-thermal effect
Go out MCF-7 tumour cells.
Beneficial effects of the present invention have：
1. cancer target peptide F3 is introduced CCMV capsid protein CP amino terminals, self group without influencing virus-like particle
Dress builds the virus-like particle with cancer target；
2.F3-CCMV virus-like particles derive from plant virus, to people without infectiousness, securely and reliably；
3. the F3-CCMV-IR780 nano particles prepared have photo-thermal curative effect to tumour cell, it can be used for biological immune
Targeted therapy.
Description of the drawings：
Fig. 1 is the agarose gel electrophoresis result of fusion F3-CP (Δ N26)；Wherein, A) fusion F3-CP (Δs
N26 PCR amplification) is as a result, using precious biology 100bp DNA standards；B) recombinant plasmid pPICZ A-F3-CP (Δ N26)
EcoRI and XhoI double digestions are as a result, using the precious biology 1kb DNA standards in Dalian；
Fig. 2 is the PAGE gel electrophoresis result of recombinant protein F3-CP；Wherein, A) difference Pichia pastoris positive transformants
The recombinant protein F3-CP of strain；B) the purification result of recombinant protein F3-CP；Using DokDo-MARKTMExtensive protein standard；
Fig. 3 is the qualification result of F3-CCMV virus-like particles；Wherein A) F3-CCMV virus-like particles electron microscope, diameter
About 18nm, insertion portion are the wild type CCMV of a diameter of 30nm, unit scaling 200nm；B) F3-CCMV virus-like particles
Dynamic scattering analysis, particle size are 18.2 ± 1.4nm；
Fig. 4 is the FPLC separating resultings of F3-CCMV-IR780 nano particles；Insertion portion is the F3-CCMV- prepared
IR780, CCMV-IR780 and free IR780 samples (from left to right)；
Fig. 5 is external intake result of the F3-CCMV-IR780 nano particles in MCF-7 tumour cells；Confocal microscopy view
Red fluorescence represents the excitation of IR780 iodide as in；Wherein, A) CCMV-IR780 processing；B) F3-CCMV-IR780 processing；
C) dissociate IR780 (ethanol solution) controls；
Fig. 6 is cell viability testing result of the F3-CCMV-IR780 nano particles to MCF-7 tumour cells；Wherein, A) no
The photo-thermal effect induced with processing；B) the microscope inspection that different disposal induces；It is followed successively by PBS from left to right, IR780 (second of dissociating
Alcoholic solution), CCMV-IR780 and F3-CCMV-IR780.
Specific implementation mode：
In order to clarify the technical characteristics of the invention, below by a specific implementation mode, and combine specification attached
Figure attached drawing, is illustrated the present invention.
The structure of 1 recombinant expression plasmid pPICZ A-F3-CP (Δ N26) of embodiment
Cancer target peptide F3 genes are inserted into the CCMV for knocking out amino terminal 1-26 amino acid residues by PCR method
In CP (Δ N26) gene, gene fusion construct F3-CP (Δ N26).The 1-25 amino acid residues of CCMV CP amino terminals are RNA
Binding structural domain, research shows that it is knocked out or replacement has no effect on CP dimers and is assembled into VLPs.First, designing two groups has mutually
The DNA oligonucleotides Olig-1 of complementary series and Olig-C1, Olig-2 and Olig-C2, respectively annealing obtain F3 genes and CP (Δs
N26) segment has 50 overlapping bases (table 1) between the two.Then, respectively with corresponding primer (F3-Up and F3-Dn, CP-Up with
CP-Dn dual asymmetric PCR (DA-PCR) and two step gene chemical synthesis of Overlap extension PCR (OE-PCR)) are carried out, using between the two
Base is overlapped as bridging.Finally, fusion is obtained by general PCR amplification with two external primers F3-Up and CP-Dn
F3-CP (Δ N26), 585 base of overall length, 195 amino acid of coding (as shown in Figure of description Figure 1A).
The oligonucleotides and primer sequence of 1 gene fusion construct F3-CP (Δ N26) of table
195 amino acid sequences are as follows：
Fusion F3-CP (Δ N26) obtained above is inserted into Pichia pastoris shuttle vector pPICZ A (to be purchased from
Invitrogen companies), structure recombinant expression plasmid pPICZ A-F3-CP (Δ N26) are reflected using EcoRI and XhoI double digestions
Its fixed molecular size range, obtains the expection band of 3.3kb and 600bp (see Figure of description Figure 1B).
The induced expression of embodiment 2F3-CCMV virus-like particles and purifying
After recombinant plasmid pPICZ A-F3-CP (Δ N26) is linearized with SacI single endonuclease digestions, is converted and finished using electroporation
Red yeast GS115.Pichia pastoris after conversion is coated with YPD tablets (1% yeast containing 100 μ g/mL bleomycins Zeocin
Cream, 2% peptone, 2% glucose, 2% agar powder), after 30 DEG C are inverted culture 2-3 days, screen positive transformants.
The positive transformants inoculation buffering glycerol complex medium BMGY of identification and buffering methanol complex media are carried out
Methanol induction.Yeast cells is collected by centrifugation, isometric 0.5mm pickling glass pearl (being purchased from Sigma companies) and 5 times of volumes are added
Disruption buffer (pH 5.5,50mM sodium phosphate, 1mM PMSF, 1mM EDTA, 5% glycerine), whirlpool concussion is broken.Centrifugation is received
Collect supernatant, carries out proteins gel electrophoresis (SDS-PAGE), the expression (as shown in Figure 2 A) of detection recombinant protein F3-CP.
By polyethylene glycol PEG sedimentations and cesium chloride density gradient centrifugation purification of recombinant proteins F3-CP, F3- is collected
CCMV virus-like particles are resuspended to assembling buffer solution (pH 5.2,100mM sodium acetate, 1mM EDTA, 5mM DTT, 0.5mM
PMSF in), SDS-PAGE detects purification effect (see Fig. 2 B).
The identification of embodiment 3F3-CCMV virus-like particles
To F3-CCMV virus-like particles after purification at transmission electron microscope TEM (nickel screen, 2% uranyl acetate negative staining) into
Row morphologic observation, the results showed that F3-CCMV is the spherical nanoparticle of diameter about 18nm, is less than wild type CCMV virions
(as shown in Figure 3A).It is 18.2 ± 1.4nm that dynamic light scattering DLS, which measures its hydrodynamic diameter, has good monodisperse
Property (see Fig. 3 B).This is with CCMV capsid proteins CP in the prior art by histidine tag (His6) and Elastin like polypeptides
(ELP) the virus-like particle size obtained after modifying is similar, should be the pseudovirion that (T=2) is made of 120 CP subunits,
Rather than the virion (T=3) that wild CCMV is made of 180 CP subunits.
The preparation of embodiment 4F3-CCMV-IR780 nano particles
F3-CCMV virus-like particles are placed in depolymerization buffer solution (pH7.5,1M sodium chloride, 20mM Tris-HCl, 5mM
DTT, 0.5mM PMSF) dialysis, 3mg/mL IR780 iodide solutions (being dissolved in ethyl alcohol) are added in gentle agitation, with assembling buffering
Liquid is dialysed again to form virus-like particle.It is detached with FPLC after ultrafiltration centrifugal concentrating, obtains F3-CCMV-IR780 nanometers
Grain (as shown in Figure 4).
Embodiment 5F3-CCMV-IR780 nano particles kill the targeting of tumour cell
By the human breast carcinoma cell lines MCF-7 of exponential phase with 2 × 104Cells/well is seeded on 8 orifice plates, and F3- is added
CCMV-IR780 nano particles are incubated 2 hours, and F3-CCMV- is observed by confocal laser scanning microscope, CLSM after fixed dyeing
IR780 is control with CCMV-IR780 and free IR780 (ethanol solution) to the targeting effect of tumour cell.As a result see
Red fluorescence is remarkably reinforced around the MCF-7 cytoplasm of Figure of description Fig. 5, F3-CCMV-IR780 processing, shows F3-CCMV-
IR780 has apparent cellular uptake and targeting binding ability to tumour cell MCF-7.
By the MCF-7 cells of exponential phase with 4 × 103Cells/well is seeded on 96 orifice plates, after 37 DEG C are cultivated 24 hours
Instead of to contain the culture medium of F3-CCMV-IR780, CCMV-IR780, free IR780 (ethanol solution) or PBS, being incubated 2 hours
After wash twice, with 850nm laser (0.6W/cm2) irradiating cell 5 minutes is to carry out photothermal treatment, after 37 DEG C are incubated 12 hours
Carry out CCK-8 cell viability detections.The result shows that F3-CCMV-IR780 has significant photo-thermal curative effect (as schemed in MCF-7 cells
Shown in 6A), microscope inspection is basic without coloring it has also been found that the major part MCF-7 cell deaths under NIR excitations (see Fig. 6 B).This
Demonstrate F3-CCMV-IR780 nano particles has good targeting and photo-thermal effect to MCF-7 tumour cells.
The above is only a preferred embodiment of the present invention, essential scope of the those of ordinary skill in the art in the present invention
The variations, modifications, additions or substitutions inside made should also belong to the scope of protection of the present invention.
Sequence table
<110>Shandong Scientific Research Academy ecological Studies institute
<120>Structure, expression and its application of the cowpea sheding green mottled virus sample particle of cancer target peptide F3 modifications
<150> 2018100118586
<151> 2018-01-05
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 108
<212> DNA
<213>Artificial synthesized (artificially synthesized)
<400> 1
catggtgcca aggtgaagga cgaaccacag agaagatcgg caaggttatc tgctaaacct 60
gctcctccaa agccagagcc caagcctaaa aaggcccctg caaagaag 108
<210> 2
<211> 108
<212> DNA
<213>Artificial synthesized (artificially synthesized)
<400> 2
ccgccacggt tccacttcct gcttggtgtc tcttctagcc gttccaatag acgatttgga 60
cgaggaggtt tcggtctcgg gttcggattt ttccggggac gtttcttc 108
<210> 3
<211> 108
<212> DNA
<213>Artificial synthesized (artificially synthesized)
<400> 3
gacgaggagg tttcggtctc gggttcggat ttttccgggg acgtttcttc gtggtccaac 60
ctgttattgt agaacccatc gcttcaggcc aaggcaaggc tattaaag 108
<210> 4
<211> 108
<212> DNA
<213>Artificial synthesized (artificially synthesized)
<400> 4
ctgctcctcc aaagccagag cccaagccta aaaaggcccc tgcaaagaag caccaggttg 60
gacaataaca tcttgggtag cgaagtccgg ttccgttccg ataatttc 108
<210> 5
<211> 30
<212> DNA
<213>Artificial synthesized (artificially synthesized)
<400> 5
ccggaattca tgggtgccaa ggtgaaggac 30
<210> 6
<211> 21
<212> DNA
<213>Artificial synthesized (artificially synthesized)
<400> 6
ctttaatagc cttgccttgg c 21
<210> 7
<211> 27
<212> DNA
<213>Artificial synthesized (artificially synthesized)
<400> 7
gtggtccaac ctgttattgt agaaccc 27
<210> 8
<211> 30
<212> DNA
<213>Artificial synthesized (artificially synthesized)
<400> 8
tgcctcgagt taatacactg gagtgaaaga 30
<210> 9
<211> 195
<212> PRT
<213>Artificial synthesized (artificially synthesized)
<400> 9
Lys Asp Glu Pro Gln Arg Arg Ser Ala Arg Leu Ser Ala Lys Pro Ala
1 5 10 15
Pro Pro Lys Pro Glu Pro Lys Pro Lys Lys Ala Pro Ala Lys Lys Val
20 25 30
Val Gln Pro Val Ile Val Glu Pro Ile Ala Ser Gly Gln Gly Lys Ala
35 40 45
Ile Lys Ala Trp Thr Gly Tyr Ser Val Ser Lys Trp Thr Ala Ser Cys
50 55 60
Ala Ala Ala Glu Ala Lys Val Thr Ser Ala Ile Thr Ile Ser Leu Pro
65 70 75 80
Asn Glu Leu Ser Ser Glu Arg Asn Lys Gln Leu Lys Val Gly Arg Val
85 90 95
Leu Leu Trp Leu Gly Leu Leu Pro Ser Val Ser Gly Thr Val Lys Ser
100 105 110
Cys Val Thr Glu Thr Gln Thr Thr Ala Ala Ala Ser Phe Gln Val Ala
115 120 125
Leu Ala Val Ala Asp Asn Ser Lys Asp Val Val Ala Ala Met Tyr Pro
130 135 140
Glu Ala Phe Lys Gly Ile Thr Leu Glu Gln Leu Thr Ala Asp Leu Thr
145 150 155 160
Ile Tyr Leu Tyr Ser Ser Ala Ala Leu Thr Glu Gly Asp Val Ile Val
165 170 175
His Leu Glu Val Glu His Val Arg Pro Thr Phe Asp Asp Ser Phe Thr
180 185 190
Pro Val Tyr
195
Claims (7)
1. a kind of cowpea sheding green mottled virus sample particle of cancer target peptide modification, which is characterized in that CCMV capsid proteins CP strikes
Except amino terminal 1-26 amino acid residues, it is inserted into cancer target peptide F3, builds F3-CP fusions, obtains F3-CCMV virus-likes
Particle.
2. the structure of the cowpea sheding green mottled virus sample particle of cancer target peptide modification as described in claim 1, feature exist
In, the synthesis of fusion F3-CP (Δ N26) is carried out using dual asymmetric PCR and Overlap extension PCR two-step PCR, wherein
F3 genes are inserted into CCMV CP (Δ N26) gene that the ends N- knock out 1-26 amino acid residues.
3. the expression of the cowpea sheding green mottled virus sample particle of cancer target peptide modification as described in claim 1, feature exist
In, the specific steps are：
(1) structure of fusion F3-CP (Δ N26)
Fusion F3-CP is carried out using dual asymmetric PCR (DA-PCR) and Overlap extension PCR (OE-PCR) two-step PCR
The synthesis of (Δ N26), wherein F3 genes are inserted into CCMV CP (Δ N26) gene that the ends N- knock out 1-26 amino acid residues.
(2) structure of recombinant expression plasmid pPICZ A-F3-CP (Δ N26)
Fusion F3-CP (Δ N26) is inserted on Pichia pastoris shuttle vector pPICZ A by digestion, connection, is weighed
Group expression plasmid pPICZ A-F3-CP (Δ N26).
(3) conversion, screening of Pichia pastoris
Pichia pastoris, culture, screening positive transformants are converted after recombinant plasmid pPICZ A-F3-CP (Δ N26) is linearized.
(4) induced expression of recombinant protein F3-CP
Methanol induction is carried out to Pichia pastoris positive transformants, thalline were collected by centrifugation, is detected by proteins gel electrophoresis after broken wall
The expression of F3-CP.
(5) purifying of recombinant protein F3-CP
Purification of recombinant proteins F3-CP is resuspended to assembling buffer solution to obtain F3-CCMV virus-like particles.
(6) identification of F3-CCMV virus-like particles
The F3-CCMV virus-like particles of acquisition are detected into its form and particle size.
4. the expression of the cowpea sheding green mottled virus sample particle of cancer target peptide modification according to claim 3, feature
It is, the Pichia pastoris described in step (3) is Pichia pastoris GS115, and condition of culture is the YPD containing bleomycin Zeocin
Tablet.
5. the expression of the cowpea sheding green mottled virus sample particle of cancer target peptide modification according to claim 3, feature
It is, polyethylene glycol PEG sedimentations and cesium chloride density gradient centrifugation purification of recombinant proteins F3-CP is utilized in step (5).
6. the cowpea sheding green mottled virus sample particle of cancer target peptide modification as described in claim 1 is identifying and is killing MCF-
Application in 7 tumour cells.
7. application as claimed in claim 6, which is characterized in that include the following steps：
(1) preparation of F3-CCMV-IR780 nano particles
IR780 iodide are contained with F3-CCMV virus-like particles, is detached through ultrafiltration centrifugation, FPLC, prepares F3-CCMV-IR780
Nano particle；
(2) the cell in vitro intake of F3-CCMV-IR780 nano particles
Using human breast carcinoma cell lines MCF-7 as indicator cells, external intake of the F3-CCMV-IR780 nano particles to tumour cell
With targeting binding ability；
(3) cell viability detects
The near-infrared effect of IR780 is induced by laser irradiation, F3-CCMV-IR780 nano particles are killed using photo-thermal effect
MCF-7 tumour cells.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810011858 | 2018-01-05 | ||
| CN2018100118586 | 2018-01-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108558990A true CN108558990A (en) | 2018-09-21 |
Family
ID=63537334
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810396256.7A Pending CN108558990A (en) | 2018-01-05 | 2018-04-28 | Structure, expression and its application of the cowpea sheding green mottled virus sample particle of cancer target peptide F3 modifications |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108558990A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111671921A (en) * | 2020-07-01 | 2020-09-18 | 中国科学院武汉病毒研究所 | Cell marking method and application thereof in rare cell MRI imaging |
| CN112285081A (en) * | 2020-10-28 | 2021-01-29 | 上海睿钰生物科技有限公司 | Method for detecting cell killing efficacy and application thereof |
| CN115747170A (en) * | 2022-08-29 | 2023-03-07 | 四川大学 | Cowpea chlorotic mottle virus-polypeptide complex and application thereof in osteoporosis treatment |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101287489A (en) * | 2005-06-01 | 2008-10-15 | 陶氏环球技术公司 | Production of multivalent virus like particles |
| CN101784655A (en) * | 2007-04-27 | 2010-07-21 | 菲尼克斯股份有限公司 | Improved production and in vivo assembly of soluble recombinant icosahedral virus-like particles |
| CN105194681A (en) * | 2015-09-17 | 2015-12-30 | 哈尔滨医科大学 | Nanometer medicine delivery system based on PPV virus-like particles and preparation method and application of nanometer medicine delivery system |
-
2018
- 2018-04-28 CN CN201810396256.7A patent/CN108558990A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101287489A (en) * | 2005-06-01 | 2008-10-15 | 陶氏环球技术公司 | Production of multivalent virus like particles |
| CN101784655A (en) * | 2007-04-27 | 2010-07-21 | 菲尼克斯股份有限公司 | Improved production and in vivo assembly of soluble recombinant icosahedral virus-like particles |
| CN105194681A (en) * | 2015-09-17 | 2015-12-30 | 哈尔滨医科大学 | Nanometer medicine delivery system based on PPV virus-like particles and preparation method and application of nanometer medicine delivery system |
Non-Patent Citations (5)
| Title |
|---|
| KIMMO PORKKA等: "A fragment of the HMGN2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo", 《PNAS》 * |
| MARK B. VAN ELDIJK等: "Designing Two Self-Assembly Mechanisms into One Viral Capsid Protein", 《J. AM. CHEM. SOC》 * |
| YUANZHENG WU等: "Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteins in Pichia pastoris and Encapsulation of Fluorescent Myoglobin", 《MATER. RES. SOC. SYMP. PROC》 * |
| YUANZHENG WU等: "Targeted Cowpea Chlorotic Mottle Virus-based Nanoparticles with Tumor-homing Peptide F3 for Photothermal Therapy", 《BIOTECHNOLOGY AND BIOPROCESS ENGINEERING》 * |
| YUFEI MA等: "Cancer-Targeted Nanotheranostics: Recent Advances and Perspectives", 《NANOTHERANOSTICS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111671921A (en) * | 2020-07-01 | 2020-09-18 | 中国科学院武汉病毒研究所 | Cell marking method and application thereof in rare cell MRI imaging |
| CN112285081A (en) * | 2020-10-28 | 2021-01-29 | 上海睿钰生物科技有限公司 | Method for detecting cell killing efficacy and application thereof |
| CN112285081B (en) * | 2020-10-28 | 2021-11-19 | 上海睿钰生物科技有限公司 | Method for detecting cell killing efficacy and application thereof |
| CN115747170A (en) * | 2022-08-29 | 2023-03-07 | 四川大学 | Cowpea chlorotic mottle virus-polypeptide complex and application thereof in osteoporosis treatment |
| CN115747170B (en) * | 2022-08-29 | 2023-08-04 | 四川大学 | Cowpea chlorotic mottle virus-polypeptide complex and application thereof in osteoporosis treatment |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tiwari et al. | Enhanced intracellular translocation and biodistribution of gold nanoparticles functionalized with a cell-penetrating peptide (VG-21) from vesicular stomatitis virus | |
| Masarapu et al. | Physalis mottle virus-like particles as nanocarriers for imaging reagents and drugs | |
| Li et al. | Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells | |
| Chakraborti et al. | Self-assembly of ferritin: structure, biological function and potential applications in nanotechnology | |
| CN108558990A (en) | Structure, expression and its application of the cowpea sheding green mottled virus sample particle of cancer target peptide F3 modifications | |
| Yildiz et al. | Engineering of Brome mosaic virus for biomedical applications | |
| CN106906230B (en) | Recombinant drug carrier protein gene and preparation method and application thereof | |
| CN108578364A (en) | Conjugate, target tumor active oxygen response medicament-carried nano micelle and preparation method and application | |
| Yang et al. | Magainin II modified polydiacetylene micelles for cancer therapy | |
| KR20200104524A (en) | Novel cell penetrating peptides and use thereof | |
| CN103705465A (en) | Slightly acidic environment targeted polypeptide modified tumor targeted nano drug delivery system, and preparation method thereof | |
| CN109395098A (en) | A kind of cancer target imaging nano particle and preparation method thereof | |
| Wang et al. | Intracellular delivery of peptide drugs using viral nanoparticles of bacteriophage P22: covalent loading and cleavable release | |
| CN109528653A (en) | Film property vesica with gene editing function and preparation method thereof, pharmaceutical composition and purposes | |
| CN111544598B (en) | Ferritin nanoparticle loaded with Survivin double inhibitors as well as preparation method and application thereof | |
| CN107184987A (en) | A kind of nanometer polypeptide carriers of targeted integration element α v β 3 of lipoic acid modification and its preparation method and application | |
| CN104800855A (en) | Tumor-targeting fusion protein drug vector for optical imaging and photodynamic therapy | |
| CN103251942B (en) | Folic acid-adenovirus tumor-targeting complex and preparation method thereof | |
| Shukla et al. | In planta production of fluorescent filamentous plant virus-based nanoparticles | |
| CN109674764B (en) | Anti-tumor magnetic drug-loaded hybrid nano-capsule and preparation method thereof | |
| CN108147990B (en) | Membrane anchoring element and application thereof | |
| Wu et al. | Targeted cowpea chlorotic mottle virus-based nanoparticles with tumor-homing peptide F3 for photothermal therapy | |
| CN114533894A (en) | Self-assembly polypeptide medicine with mitochondrion targeting function and preparation method and application thereof | |
| CN112826943B (en) | Protein nano-carrier, carrier loaded with targeting substance, preparation method and application | |
| Yuan et al. | Virus-like particle-based nanocarriers as an emerging platform for drug delivery |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180921 |